RESUMEN
Summer maize is frequently subjected to waterlogging damage because of increased and variable rainfall during the growing season. The application of 6-benzyladenine (6-BA) can effectively mitigate the waterlogging effects on plant growth and increase the grain yield of waterlogged summer maize. However, the mechanisms underlying this process and the involvement of 6-BA in relevant signal transduction pathways remain unclear. In this study, we explored the effects of 6-BA on waterlogged summer maize using a phosphoproteomic technique to better understand the mechanism by which summer maize growth improves following waterlogging. Application of 6-BA inhibited the waterlogging-induced increase in abscisic acid (ABA) content and increased the phosphorylation levels of proteins involved in ABA signaling; accordingly, stomatal responsiveness to exogenous ABA increased. In addition, the application of 6-BA had a long-term effect on signal transduction pathways and contributed to rapid responses to subsequent stresses. Plants primed with 6-BA accumulated more ethylene and jasmonic acid in response to subsequent waterlogging; accordingly, leaf SPAD, antioxidase activity, and root traits improved by 6-BA priming. These results suggest that the effects of 6-BA on hormone signal transduction pathways are anamnestic, which enables plants to show faster or stronger defense responses to stress.
Asunto(s)
Compuestos de Bencilo , Reguladores del Crecimiento de las Plantas , Purinas , Agua , Zea mays , Compuestos de Bencilo/metabolismo , Reguladores del Crecimiento de las Plantas/fisiología , Purinas/metabolismo , Estaciones del Año , Transducción de Señal , Agua/metabolismo , Zea mays/fisiologíaRESUMEN
Callogenesis, the process during which explants derived from differentiated plant tissues are subjected to a trans-differentiation step characterized by the proliferation of a mass of cells, is fundamental to indirect organogenesis and the establishment of cell suspension cultures. Therefore, understanding how callogenesis takes place is helpful to plant tissue culture, as well as to plant biotechnology and bioprocess engineering. The common herbaceous plant stinging nettle (Urtica dioica L.) is a species producing cellulosic fibres (the bast fibres) and a whole array of phytochemicals for pharmacological, nutraceutical and cosmeceutical use. Thus, it is of interest as a potential multi-purpose plant. In this study, callogenesis in internode explants of a nettle fibre clone (clone 13) was studied using RNA-Seq to understand which gene ontologies predominate at different time points. Callogenesis was induced with the plant growth regulators α-napthaleneacetic acid (NAA) and 6-benzyl aminopurine (BAP) after having determined their optimal concentrations. The process was studied over a period of 34 days, a time point at which a well-visible callus mass developed on the explants. The bioinformatic analysis of the transcriptomic dataset revealed specific gene ontologies characterizing each of the four time points investigated (0, 1, 10 and 34 days). The results show that, while the advanced stage of callogenesis is characterized by the iron deficiency response triggered by the high levels of reactive oxygen species accumulated by the proliferating cell mass, the intermediate and early phases are dominated by ontologies related to the immune response and cell wall loosening, respectively.
Asunto(s)
Desarrollo de la Planta/genética , Transcriptoma/genética , Urtica dioica/crecimiento & desarrollo , Urtica dioica/genética , Compuestos de Bencilo/metabolismo , Compuestos de Bencilo/farmacología , Técnicas de Cultivo de Célula/métodos , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Hierro/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Reguladores del Crecimiento de las Plantas/farmacología , Raíces de Plantas/crecimiento & desarrollo , Purinas/metabolismo , Purinas/farmacología , RNA-Seq/métodos , Especies Reactivas de Oxígeno/metabolismo , Urtica dioica/citología , Urtica dioica/metabolismoRESUMEN
Sphingosine-1-phosphate (S1P) is an important bioactive lipid molecule in cell membrane metabolism and binds to G protein-coupled S1P receptors (S1PRs) to regulate embryonic development, physiological homeostasis, and pathogenic processes in various organs. S1PRs are lipid-sensing receptors and are therapeutic targets for drug development, including potential treatment of COVID-19. Herein, we present five cryo-electron microscopy structures of S1PRs bound to diverse drug agonists and the heterotrimeric Gi protein. Our structural and functional assays demonstrate the different binding modes of chemically distinct agonists of S1PRs, reveal the mechanical switch that activates these receptors, and provide a framework for understanding ligand selectivity and G protein coupling.
Asunto(s)
Receptores de Esfingosina-1-Fosfato/agonistas , Azetidinas/química , Azetidinas/metabolismo , Compuestos de Bencilo/química , Compuestos de Bencilo/metabolismo , Microscopía por Crioelectrón , Humanos , Simulación de Dinámica Molecular , Unión Proteica , Estructura Cuaternaria de Proteína , Transducción de Señal , Receptores de Esfingosina-1-Fosfato/genética , Receptores de Esfingosina-1-Fosfato/metabolismoRESUMEN
We report the design of a new model based on a small neutral 8-aryl-3-formylBODIPY and its suitability to develop privileged highly bright and photostable fluorescent probes for selective and, more importantly, covalent staining of mitochondria.
Asunto(s)
Compuestos de Boro/química , Colorantes Fluorescentes/química , Mitocondrias/química , Imagen Óptica , Compuestos de Bencilo/química , Compuestos de Bencilo/metabolismo , Compuestos de Boro/síntesis química , Colorantes Fluorescentes/síntesis química , Humanos , Mitocondrias/metabolismo , Estructura Molecular , Células PC-3 , Procesos FotoquímicosRESUMEN
The toxicological manifestation of many pollutants relies upon their binding to the aryl hydrocarbon receptor (AHR), and it follows a cascade of reactions culminating in an elevated expression of cytochrome P450 (CYP) 1 enzymes. CYP1A1 and CYP1B1 are associated with enhanced carcinogenesis when chronically exposed to certain polyaromatic hydrocarbons, and their inhibition may lead to chemoprevention. We evaluated dibenzyl trisulfide (DTS), expressed in the ethnomedical plant, Petiveria alliacea, for such potential chemoprevention. Using recombinant human CYP1A1 and CYP1B1 bactosomes on a fluorogenic assay, we first demonstrated that DTS moderately inhibited both enzymes with half maximal inhibitory concentration (IC50) values of 1.3 ± 0.3 and 1.7 ± 0.3 µM, respectively. Against CYP1A1, DTS was a reversible, competitive inhibitor with an apparent inhibitory constant (Ki) of 4.55 ± 0.37 µM. In silico molecular modeling showed that DTS binds with an affinity of -39.8 kJ·mol-1, situated inside the binding pocket, approximately 4.3 Å away from the heme group, exhibiting interactions with phenylalanine residue 123 (Phe-123), Phe-224, and Phe-258. Lastly, zebrafish (Danio rerio) embryos were exposed to 0.08-0.8 µM DTS from 24 to 96 h post fertilization (hpf) with the in vivo ethoxyresorufin-O-deethylase (EROD) assay, and, at 96 hpf, DTS significantly suppressed EROD CYP1A activity in a dose-dependent manner, with up to 60% suppression in the highest 0.8 µM exposure group. DTS had no impact on gene transcription levels for cyp1a and aryl hydrocarbon receptor 2 (ahr2). In co-exposure experiments, DTS suppressed CYP1A activity induced by both B[a]P and PCB-126, although these reductions were not significant. Taken together, these results demonstrate that DTS is a direct, reversible, competitive inhibitor of the carcinogen-activating CYP1A enzyme, binding in the active site pocket close to the heme site, and shows potential in chemoprevention.
Asunto(s)
Compuestos de Bencilo/farmacología , Citocromo P-450 CYP1A1/antagonistas & inhibidores , Citocromo P-450 CYP1B1/antagonistas & inhibidores , Inhibidores Enzimáticos del Citocromo P-450/farmacología , Receptores de Hidrocarburo de Aril/metabolismo , Sulfuros/farmacología , Proteínas de Pez Cebra/metabolismo , Activación Metabólica , Animales , Benzo(a)pireno/metabolismo , Benzo(a)pireno/toxicidad , Compuestos de Bencilo/metabolismo , Sitios de Unión , Unión Competitiva , Dominio Catalítico , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Inhibidores Enzimáticos del Citocromo P-450/metabolismo , Regulación de la Expresión Génica , Humanos , Bifenilos Policlorados/metabolismo , Bifenilos Policlorados/toxicidad , Unión Proteica , Receptores de Hidrocarburo de Aril/genética , Sulfuros/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
6-benzylaminopurine (6-BA) is one of the first synthetic hormones and has been widely used in fruit cultivation, gardening and agriculture. However, excessive use of 6-BA will cause potential harm to the environment and humans. Therefore, our research focused on assessing the impact of 6-BA on the development and neurobehavior of zebrafish. The results showed that 6-BA had little effect on the embryos from 2 hpf to 10 hpf. However, delayed development, decreased survival and hatchability were observed under 30 and 40 mg/L 6-BA from 24 hpf. 6-BA also reduced surface tension of embryonic chorions at 24 hpf. In addition, 6-BA caused abnormal morphology and promoted the accumulation of oxidative stress. Transcription of genes in connection with development and oxidative stress was also strikingly altered. Results of movement assay showed that zebrafish were less active and their behavior was significantly inhibited under the 20 and 30 mg/L 6-BA treatments. Locomotion-related genes th and mao were down-regulated by gradient, while the transcription of dbh was upregulated at a low concentration (2 mg/L) but decreased as the concentration increased. Moreover, 6-BA exposure caused increased arousal and decreased sleep. Sleep/wake related genes hcrt and hcrtr2 were upregulated, but decreased at 30 mg/L, while the mRNA level of aanat2 was reduced in a concentration-dependent manner. To sum up, our results showed that 6-BA induced developmental toxicity, promoted the accumulation of oxidative stress, and damaged locomotion and sleep/wake behavior.
Asunto(s)
Contaminantes Químicos del Agua , Pez Cebra , Animales , Compuestos de Bencilo/metabolismo , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Humanos , Receptores de Orexina/metabolismo , Estrés Oxidativo , Purinas/metabolismo , Contaminantes Químicos del Agua/metabolismoRESUMEN
Flavin-containing monooxygenase (FMO) catalyzes the oxygenation of a wide variety of medicines and dietary-derived compounds. However, little information is available regarding drug interactions mediated by FMO3 in vivo. Consequently, we investigated interactions between FMO substrates in humanized-liver mice. Trimethylamine-d9 and itopride were, respectively, intravenously and orally administered to humanized-liver mice (n = 5-7). The pharmacokinetic profiles of itopride (the victim drug) in the presence of trimethylamine (the perpetrator drug) were determined for 24 h after co-administration using liquid chromatography/tandem mass spectrometry. Itopride (10 mg/kg) was extensively oxygenated in humanized-liver mice to its N-oxide. The plasma concentrations of itopride N-oxide after co-administration of itopride and trimethylamine (10 and 100 mg/kg) were significantly suppressed in a dose-dependent manner, but only during the early phase, i.e., up to 2 h after co-administration. With the higher dose of trimethylamine, the areas under the concentration-time curves of itopride and its N-oxide significantly increased (1.6-fold) and decreased (to 60%), respectively; modeling suggested that these modified pharmacokinetics resulted from suppression of the in vivo hepatic intrinsic clearance (to 67%). These results suggest that food-derived trimethylamine may result in interactions with FMO drug substrates immediately after administration; however, the potential for this to occur in vivo may be limited.
Asunto(s)
Benzamidas/metabolismo , Compuestos de Bencilo/metabolismo , Hígado/metabolismo , Metilaminas/metabolismo , Oxigenasas/metabolismo , Animales , Benzamidas/química , Benzamidas/farmacocinética , Compuestos de Bencilo/química , Compuestos de Bencilo/farmacocinética , Interacciones Farmacológicas , Hígado/química , Masculino , Metilaminas/química , Metilaminas/farmacocinética , Ratones , Ratones Transgénicos , Oxigenasas/químicaRESUMEN
BACKGROUND: The latest coronavirus SARS-CoV-2, discovered in China and rapidly spread Worldwide. COVID-19 affected millions of people and killed hundreds of thousands worldwide. There are many ongoing studies investigating drug(s) suitable for preventing and/or treating this pandemic; however, there are no specific drugs or vaccines available to treat or prevent SARS-CoV-2 as of today. METHODS: Fifty-eight fragrance materials, which are classified as allergen fragrance molecules, were selected and used in this study. Docking simulations were carried out using four functional proteins; the Covid19 Main Protase (MPro), Receptor binding domain (RBD) of spike protein, Nucleocapsid, and host Bromodomain protein (BRD2), as target macromolecules. Three different software, AutoDock, AutoDock Vina (Vina), and Molegro Virtual Docker (MVD), running a total of four different docking protocol with optimized energy functions were used. Results were compared with the five molecules reported in the literature as potential drugs against COVID-19. Virtual screening was carried out using Vina, molecules satisfying our cut-off (- 6.5 kcal/mol) binding affinity was confirmed by MVD. Selected molecules were analyzed using the flexible docking protocol of Vina and AutoDock default settings. RESULTS: Ten out of 58 allergen fragrance molecules were selected for further docking studies. MPro and BRD2 are potential targets for the tested allergen fragrance molecules, while RBD and Nucleocapsid showed weak binding energies. According to AutoDock results, three molecules, Benzyl Cinnamate, Dihydroambrettolide, and Galaxolide, had good binding affinities to BRD2. While Dihydroambrettolide and Galaxolide showed the potential to bind to MPro, Sclareol and Vertofix had the best calculated binding affinities to this target. When the flexible docking results analyzed, all the molecules tested had better calculated binding affinities as expected. Benzyl Benzoate and Benzyl Salicylate showed good binding affinities to BRD2. In the case of MPro, Sclareol had the lowest binding affinity among all the tested allergen fragrance molecules. CONCLUSION: Allergen fragrance molecules are readily available, cost-efficient, and shown to be safe for human use. Results showed that several of these molecules had comparable binding affinities as the potential drug molecules reported in the literature to target proteins. Thus, these allergen molecules at correct doses could have significant health benefits.
Asunto(s)
Alérgenos/química , Alérgenos/inmunología , Tratamiento Farmacológico de COVID-19 , COVID-19/inmunología , Odorantes , Perfumes/química , SARS-CoV-2/química , SARS-CoV-2/inmunología , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/química , Adenosina Monofosfato/metabolismo , Alanina/análogos & derivados , Alanina/química , Alanina/metabolismo , Alérgenos/administración & dosificación , Alérgenos/uso terapéutico , Benzopiranos/química , Benzopiranos/metabolismo , Compuestos de Bencilo/química , Compuestos de Bencilo/metabolismo , Cinamatos/química , Cinamatos/metabolismo , Proteasas 3C de Coronavirus/química , Proteasas 3C de Coronavirus/metabolismo , Proteínas de la Nucleocápside de Coronavirus/química , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Diterpenos/química , Diterpenos/metabolismo , Evaluación Preclínica de Medicamentos , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Perfumes/administración & dosificación , Perfumes/uso terapéutico , Fosfoproteínas/química , Fosfoproteínas/metabolismo , SARS-CoV-2/efectos de los fármacos , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Transcription is an essential biological process in bacteria requiring a core enzyme, RNA polymerase (RNAP). Bacterial RNAP is catalytically active but requires sigma (σ) factors for transcription of natural DNA templates. σ factor binds to RNAP to form a holoenzyme which specifically recognizes a promoter, melts the DNA duplex, and commences RNA synthesis. Inhibiting the binding of σ to RNAP is expected to inhibit bacterial transcription and growth. We previously identified a triaryl hit compound that mimics σ at its major binding site of RNAP, thereby inhibiting the RNAP holoenzyme formation. In this study, we modified this scaffold to provide a series of benzyl and benzoyl benzoic acid derivatives possessing improved antimicrobial activity. A representative compound demonstrated excellent activity against Staphylococcus epidermidis with minimum inhibitory concentrations reduced to 0.5 µg/mL, matching that of vancomycin. The molecular mechanism of inhibition was confirmed using biochemical and cellular assays. Low cytotoxicity and metabolic stability of compounds demonstrated the potential for further studies.
Asunto(s)
Proteínas Bacterianas/metabolismo , Benzoatos/farmacología , Benzofenonas/farmacología , Compuestos de Bencilo/farmacología , ARN Polimerasas Dirigidas por ADN/metabolismo , Factor sigma/metabolismo , Animales , Bacterias/efectos de los fármacos , Benzoatos/síntesis química , Benzoatos/metabolismo , Benzofenonas/síntesis química , Benzofenonas/metabolismo , Compuestos de Bencilo/síntesis química , Compuestos de Bencilo/metabolismo , Pruebas de Sensibilidad Microbiana , Microsomas Hepáticos/metabolismo , Unión Proteica/efectos de los fármacos , RatasRESUMEN
This is the first report on identification and quantification of important hepatoprotective and anticancer polyphenolic lignans such as phyllanthin (PH), hypophyllanthin (HPH), niranthin (NH) and phyltetralin (PT) in natural plant and in vitro cultures of Phyllanthus tenellus Roxb. The identification of lignans was carried out by Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) and quantified using High-Performance Liquid Chromatography (HPLC). In addition, an efficient protocol has been developed for multiple shoot induction in nodal explants of in vitro derived shoots of P. tenellus. Maximum number of shoot regeneration (7.83 ± 0.15) was achieved on medium incorporated with 1.0 mg/l 6-Benzylaminopurine (BAP). The medium containing Indole-3-acetic acid (IAA) 2 mg/l was superior for induction of rooting in in vitro raised shoots. The plantlets were acclimatized to the field condition with 100% survival. The quantitative HPLC analysis showed that the lignan content was variable with the auxins and cytokinins incorporated in the medium. The lignan content was higher in callus grown on Murashige and Skoog (MS) medium + 2.0 mg/l Naphthaleneacetic acid (NAA). The reported protocol can be used for mass propagation and application of biotechnological approaches for improvement of P. tenellus. The results indicate intriguing possibilities for the utilization of P. tenellus plant parts as an alternative source and of callus culture to scale up bioactive lignan production for pharmaceutical applications.
Asunto(s)
Lignanos/metabolismo , Phyllanthus/metabolismo , Compuestos de Bencilo/metabolismo , Medios de Cultivo/metabolismo , Citocininas/metabolismo , Ácidos Indolacéticos/metabolismo , Raíces de Plantas/metabolismo , Brotes de la Planta/metabolismo , Purinas/metabolismoRESUMEN
There has been controversy over the cardiovascular safety of domperidone, attributable to the lack of a well-designed study as well as inconsistent results. This study aimed to examine the risk of severe domperidone-induced ventricular arrhythmia (VA), compared to mosapride, itopride, or non-use of all three prokinetics, in the general population. We conducted a population-based, self-controlled case series analysis. Enrolled subjects were individuals who were diagnosed with severe VA and were prescribed domperidone, mosapride, or itopride from 2003 to 2013 in the National Health Insurance Service-National Sample Cohort. The incidence rate ratio for severe VA was measured during exposure to prokinetics and compared with unexposed periods and itopride (no-proarrhythmic effect)-exposure periods, as control. A total of 2,817 subjects were included. Domperidone, mosapride, or itopride use was associated with increased risk of severe VA, compared with non-use (adjusted incidence rate ratios (IRR) of 1.342 (95% CI 1.096-1.642), 1.350 (95% CI 1.105-1.650), and 1.486 (95% CI 1.196-1.845), respectively). The risk of severe domperidone-induced VA was lower, compared to that of itopride [adjusted IRR of 0.548 (95% CI 0.345-0.870)]. Of the subjects who had been prescribed all three prokinetics, domperidone-exposure was associated with a lower risk of severe VA, compared to itopride-exposure (crude IRR, 0.571; 0.358-0.912). Mosapride-exposure did not show IRR difference for severe VA, compared to itopride-exposure. Domperidone, mosapride, or itopride use is associated with an increased risk of severe VA. However, the magnitude of association was modest and domperidone use does not increase further the risk, compared with other prokinetics.
Asunto(s)
Antieméticos/efectos adversos , Arritmias Cardíacas/etiología , Domperidona/efectos adversos , Adolescente , Adulto , Anciano , Antieméticos/metabolismo , Antieméticos/uso terapéutico , Arritmias Cardíacas/epidemiología , Arritmias Cardíacas/patología , Benzamidas/efectos adversos , Benzamidas/metabolismo , Benzamidas/uso terapéutico , Compuestos de Bencilo/efectos adversos , Compuestos de Bencilo/metabolismo , Compuestos de Bencilo/uso terapéutico , Niño , Preescolar , Bases de Datos Factuales , Domperidona/metabolismo , Domperidona/uso terapéutico , Femenino , Humanos , Incidencia , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Morfolinas/efectos adversos , Morfolinas/metabolismo , Morfolinas/uso terapéutico , Factores de Riesgo , Índice de Severidad de la Enfermedad , Adulto JovenRESUMEN
The modulation of the sphingosine 1-phosphate receptor is an approved treatment for relapsing multiple sclerosis because of its anti-inflammatory effect of retaining lymphocytes in lymph nodes. Different sphingosine 1-phosphate receptor subtypes are expressed in the brain and spinal cord, and their pharmacological effects may improve disease development and neuropathology. Siponimod (BAF312) is a novel sphingosine 1-phosphate receptor modulator that has recently been approved for the treatment of active secondary progressive multiple sclerosis (MS). In this review article, we summarize recent evidence suggesting that the active role of siponimod in patients with progressive MS may be due to direct interaction with central nervous system cells. Additionally, we tried to summarize our current understanding of the function of siponimod and discuss the effects observed in the case of MS.
Asunto(s)
Azetidinas/metabolismo , Compuestos de Bencilo/metabolismo , Sistema Nervioso Central/fisiopatología , Esclerosis Múltiple/tratamiento farmacológico , Moduladores de los Receptores de fosfatos y esfingosina 1/metabolismo , Animales , Modelos Animales de Enfermedad , Humanos , RatonesRESUMEN
Lipase from Thermomyces lanuginosus (TLL) was immobilized onto a novel heterofunctional support, divinyl sulfone (DVS) superparamagnetic nanoparticles (SPMNs) functionalized with polyethyleneimine (PEI). Particle size and zeta potential measurements, elemental analysis, X-ray powder diffraction, magnetic measurements, and infrared spectroscopy analysis were used to characterize the TLL preparations. At pH 10, it was possible to achieve 100 % of immobilization yield in 1â¯h. The immobilization pH gives TLL preparations with different stabilities; indeed the TLL preparation immobilized at pH 5.0 was the most stable during the thermal inactivation at all pH values. For the hydrolysis of racemic methyl mandelate, the nanobiocatalysts immobilized at pH 5.0 and blocked with ethylenediamine (EDA) and ethanolamine (ETA) obtained good enantioselectivities (68 % and 72 %, respectively) with high catalytic activities in the reaction medium at pH 7.0. The operational stability of the systems was evaluated in the esterification reaction of benzyl alcohol, obtaining up to 61 % conversion after the seventh reaction cycle. These results show that SPMN@PEI-DVS support is a robust strategy for the easy and rapid recovery of the nanobiocatalyst by applying a magnetic field, showing great potential for industrial applications.
Asunto(s)
Enzimas Inmovilizadas/química , Eurotiales/enzimología , Lipasa/química , Nanopartículas Magnéticas de Óxido de Hierro/química , Polietileneimina/química , Sulfonas/química , Compuestos de Bencilo/metabolismo , Estabilidad de Enzimas , Enzimas Inmovilizadas/metabolismo , Esterificación , Etanolamina/química , Etilenodiaminas/química , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Concentración de Iones de Hidrógeno , Hidrólisis , Lipasa/metabolismo , Temperatura , Factores de TiempoRESUMEN
Spinach and Broccoli are fluorogenic RNA aptamers that bind DFHBI, a mimic of the chromophore in green fluorescent protein, and activate its fluorescence. Spinach/Broccoli-DFHBI complexes exhibit high fluorescence in vitro, but they exhibit lower fluorescence in mammalian cells. Here, computational screening was used to identify BI, a DFHBI derivative that binds Broccoli with higher affinity and leads to markedly higher fluorescence in cells compared to previous ligands. BI prevents thermal unfolding of Broccoli at 37 °C, leading to more folded Broccoli and thus more fluorescent Broccoli-BI complexes in cells. Broccoli-BI complexes are more photostable owing to impaired photoisomerization and rapid unbinding of photoisomerized cis-BI. These properties enable single mRNA containing 24 Broccoli aptamers to be imaged in live mammalian cells treated with BI. Small molecule ligands can thus promote RNA folding in cells, and thus allow single mRNA imaging with fluorogenic aptamers.
Asunto(s)
Aptámeros de Nucleótidos/química , Compuestos de Bencilo/química , Brassica/genética , Colorantes Fluorescentes/química , Imidazolinas/química , ARN Mensajero/química , Aptámeros de Nucleótidos/metabolismo , Compuestos de Bencilo/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Imidazolinas/metabolismo , Isomerismo , Imagen Óptica , Procesos Fotoquímicos , Pliegue del ARN , Imagen Individual de Molécula , Temperatura de TransiciónAsunto(s)
Azetidinas/metabolismo , Azetidinas/uso terapéutico , Compuestos de Bencilo/metabolismo , Compuestos de Bencilo/uso terapéutico , Negro o Afroamericano/genética , Citocromo P-450 CYP2C9/genética , Esclerosis Múltiple/etnología , Moduladores de los Receptores de fosfatos y esfingosina 1/metabolismo , Moduladores de los Receptores de fosfatos y esfingosina 1/uso terapéutico , Adulto , Negro o Afroamericano/etnología , Alelos , Ensayos Clínicos como Asunto , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Esclerosis Múltiple/tratamiento farmacológico , PrevalenciaRESUMEN
Phenylalanine ammonia-lyase (PAL) catalyzes the reversible deamination of phenylalanine to cinnamic acid and ammonia. Algae have been considered as biofactories for PAL production, however, biochemical characterization of PAL and its potency for myristicin biotransformation into MMDA (3-methoxy-4, 5-methylenedioxyamphetamine) has not been studied yet. Thus, PAL from Anabaena flos-aquae and Spirulina platensis has been purified, comparatively characterized and its affinity to transform myristicin was assessed. The specific activity of purified PAL from S. platensis (73.9 µmol/mg/min) and A. flos-aquae (30.5 µmol/mg/min) was increased by about 2.9 and 2.4 folds by gel-filtration comparing to their corresponding crude enzymes. Under denaturing-PAGE, a single proteineous band with a molecular mass of 64 kDa appeared for A. flos-aquae and S. platensis PAL. The biochemical properties of the purified PAL from both algal isolates were determined comparatively. The optimum temperature of S. platensis and A. flos-aquae PAL for forward or reverse activity was reported at 30°C, while the optimum pH for PAL enzyme isolated from A. flos-aquae was 8.9 for forward and reverse activities, and S. platensis PAL had maximum activities at pH 8.9 and 8 for forward and reverse reactions, respectively. Luckily, the purified PALs have the affinity to hydroaminate the myristicin to MMDA successfully in one step. Furthermore, a successful method for synthesis of MMDA from myristicin in two steps was also established. Gas chromatography-mass spectrometry (GC-MS) analysis was conducted to track the product formation.
Asunto(s)
Compuestos de Bencilo/metabolismo , Dioxolanos/metabolismo , Dolichospermum flos-aquae/enzimología , Fenilanina Amoníaco-Liasa/aislamiento & purificación , Fenilanina Amoníaco-Liasa/metabolismo , Pirogalol/análogos & derivados , 3,4-Metilenodioxianfetamina/análogos & derivados , 3,4-Metilenodioxianfetamina/metabolismo , Derivados de Alilbenceno , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Biotransformación , Concentración de Iones de Hidrógeno , Estructura Molecular , Peso Molecular , Fenilanina Amoníaco-Liasa/química , Pirogalol/metabolismo , Spirulina/enzimología , Especificidad por Sustrato , TemperaturaRESUMEN
Myristicin is widely distributed in spices and medicinal plants. The aim of this study was to explore the role of metabolic activation of myristicin in its potential toxicity through a metabolomic approach. The myristicin- N-acetylcysteine adduct was identified by comparing the metabolic maps of myristicin and 1'-hydroxymyristicin. The supplement of N-acetylcysteine could protect against the cytotoxicity of myristicin and 1'-hydroxymyristicin in primary mouse hepatocytes. When the depletion of intracellular N-acetylcysteine was pretreated with diethyl maleate in hepatocytes, the cytotoxicity induced by myristicin and 1'-hydroxymyristicin was deteriorated. It suggested that the N-acetylcysteine adduct resulting from myristicin bioactivation was closely associated with myristicin toxicity. Screening of human recombinant cytochrome P450s (CYPs) and treatment with CYP inhibitors revealed that CYP1A1 was mainly involved in the formation of 1'-hydroxymyristicin. Collectively, this study provided a global view of myristicin metabolism and identified the N-acetylcysteine adduct resulting from myristicin bioactivation, which could be used for understanding the mechanism of myristicin toxicity.
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Compuestos de Bencilo/metabolismo , Compuestos de Bencilo/toxicidad , Dioxolanos/metabolismo , Dioxolanos/toxicidad , Hepatocitos/efectos de los fármacos , Pirogalol/análogos & derivados , Acetilcisteína/química , Acetilcisteína/metabolismo , Activación Metabólica , Derivados de Alilbenceno , Animales , Compuestos de Bencilo/química , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citocromo P-450 CYP1A1/metabolismo , Dioxolanos/química , Hepatocitos/citología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Pirogalol/química , Pirogalol/metabolismo , Pirogalol/toxicidadRESUMEN
25B-NBF, 2-(4-bromo-2,5-dimethoxyphenyl)-N-(2-fluorobenzyl)ethanamine, is a new psychoactive substance classified as a phenethylamine. It is a potent agonist of the 5-hydroxytryptamine receptor, but little is known about its metabolism and elimination properties since it was discovered. To aid 25B-NBF abuse screening, the metabolic characteristics of 25B-NBF were investigated in human hepatocytes and human cDNA-expressed cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes using liquid chromatographyâ»high resolution mass spectrometry. At a hepatic extraction ratio of 0.80, 25B-NBF was extensively metabolized into 33 metabolites via hydroxylation, O-demethylation, bis-O-demethylation, N-debenzylation, glucuronidation, sulfation, and acetylation after incubation with pooled human hepatocytes. The metabolism of 25B-NBF was catalyzed by CYP1A1, CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2J2, CYP3A4, and UGT2B7 enzymes. Based on these results, it is necessary to develop a bioanalytical method for the determination of not only 25B-NBF but also its metabolites in biological samples for the screening of 25B-NBF abuse.
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Compuestos de Bencilo/química , Compuestos de Bencilo/metabolismo , Etilaminas/química , Etilaminas/metabolismo , Hepatocitos/metabolismo , Fenetilaminas/metabolismo , Antagonistas de la Serotonina/metabolismo , Biocatálisis , Cromatografía Liquida , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Evaluación Preclínica de Medicamentos , Expresión Génica , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Humanos , Estructura Molecular , Receptores de Serotonina/metabolismo , Relación Estructura-Actividad , Espectrometría de Masas en TándemRESUMEN
Siponimod, a next-generation selective sphingosine-1-phosphate receptor modulator, is currently being investigated for the treatment of secondary progressive multiple sclerosis. We investigated the absorption, distribution, metabolism, and excretion (ADME) of a single 10-mg oral dose of [14C]siponimod in four healthy men. Mass balance, blood and plasma radioactivity, and plasma siponimod concentrations were measured. Metabolite profiles were determined in plasma, urine, and feces. Metabolite structures were elucidated using mass spectrometry and comparison with reference compounds. Unchanged siponimod accounted for 57% of the total plasma radioactivity (area under the concentration-time curve), indicating substantial exposure to metabolites. Siponimod showed medium to slow absorption (median Tmax: 4 hours) and moderate distribution (Vz/F: 291 l). Siponimod was mainly cleared through biotransformation, predominantly by oxidative metabolism. The mean apparent elimination half-life of siponimod in plasma was 56.6 hours. Siponimod was excreted mostly in feces in the form of oxidative metabolites. The excretion of radioactivity was close to complete after 13 days. Based on the metabolite patterns, a phase II metabolite (M3) formed by glucuronidation of hydroxylated siponimod was the main circulating metabolite in plasma. However, in subsequent mouse ADME and clinical pharmacokinetic studies, a long-lived nonpolar metabolite (M17, cholesterol ester of siponimod) was identified as the most prominent systemic metabolite. We further conducted in vitro experiments to investigate the enzymes responsible for the oxidative metabolism of siponimod. The selective inhibitor and recombinant enzyme results identified cytochrome P450 2C9 (CYP2C9) as the predominant contributor to the human liver microsomal biotransformation of siponimod, with minor contributions from CYP3A4 and other cytochrome P450 enzymes.
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Azetidinas/metabolismo , Compuestos de Bencilo/metabolismo , Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP3A/metabolismo , Receptores de Lisoesfingolípidos/agonistas , Adolescente , Adulto , Animales , Biotransformación/fisiología , Heces , Semivida , Voluntarios Sanos , Humanos , Masculino , Ratones , Microsomas Hepáticos/metabolismo , Persona de Mediana Edad , Oxidación-Reducción , Estrés Oxidativo/fisiología , Adulto JovenRESUMEN
The biotransformation of the lignan (-)-cubebin by filamentous fungi Aspergillus terreus and Aspergillus niger is an efficient bioprocess for obtaining (-)-hinokinin and (-)-parabenzlactone. The relevance of getting (-)-hinokinin is due to its promising effect against oral pathogens, especially S. sanguinis (both MIC and MBC 12.5 µg/mL), and other previous reported effects against Chagas disease and as anti-inflammatory. The advantage of using fungal transformation is the use of non-toxic and/or non-pollutant reagents and/or solvents in comparison with semi-synthesis. Microbial transformation of (-)-cubebin is also important to evaluate its human metabolism, since Aspergillus species are capable of mimicking P450 reactions, providing possible products of the metabolism, which is important in the assessment of its efficacy and safety. Furthermore, the present study describes a reliable RP-HPLC method to perform quantification of (-)-hinokinin in fungal extracts. It is simple, fast, selective, linear, precise, accurate and robust according to validation guidelines.